EGF, epithelium and Action of serotonergic drugs on calcium level 179 Action of serotonin antagonists on cytoplasmic calcium levels in early embryos of sea urchin Lytechinus pictus YURI B. SHMUKLER 1 *, GENNADY A. BUZNIKOV 1 and MICHAEL J. WHITAKER 2 1 N.K.Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia and 2 Department of Physiological Sciences, The Medical School, University of Newcastle, Newcastle-upon-Tyne, United Kingdom ABSTRACT Possible interaction of the serotonergic system with intracellular calcium mechanisms was investigated using techniques of ratio imaging measurement of intracellular Ca 2+ and confocal microscopy in cleaving embryos of sea urchin Lytechinus pictus. Some serotonin antagonists specifically increase free intracellular Ca 2+ and evoke transient regression of the first cleavage furrow, suggesting possible linkage of serotonergic and calcium mechanisms in the regulation of cellular events during cleavage divisions. These effects were more pronounced in the experiments with hydrophilic 5-HT-antagonists, quarternary ammonium salts that do not penetrate the cell membrane. Thus, it appears that 5-HT-receptors which mediate these effects are localised on the cell membrane, whereas previously studied receptors mediating the cytostatic action of lipophilic 5-HT-antagonists are localised intracellularly. KEY WORDS: serotonin, Ca 2+ , cleavage divisions, cytoskeleton, receptor Int. J. Dev. Biol. 43: 179-182 (1999) 0214-6282/99/$15.00 © UBC Press Printed in Spain www.lg.ehu.es/ijdb *Address for reprints: N.K.Koltzov Institute of Developmental Biology, 26 Vavilov st., Moscow, 117808, Russia. FAX: (7 095) 135-3055. e-mail: ybshm@ibrran.msk.su It is well known now that neurotransmitters are multifunctional substances playing,inparticular,animportantroleinregulatoryeventsofembryogenesis, including the pre-nervous stages of development (Buznikov, 1967, 1990; Buznikov et al ., 1996). One of the most interesting peculiarities of pre- nervous neurotransmitter systems, is the co-existence of intracellular and plasma membrane neurotransmitter receptors (Buznikov, 1990; Shmukler, 1993; Buznikov et al ., 1996). Both types of receptors are functionally coupled to various second messengers, including cyclic nucleotides (Shmukler and Grigoriev, 1984; Shmukler et al ., 1986; Capasso et al ., 1988), phosphoinositides (Buznikov et al ., 1993), and Ca 2+ (Shmukler et al ., 1986; Buznikov et al ., 1993, 1996, 1997). The present work is devoted to the study of the direct effects of drugs related to one pre-nervous neurotransmitter, 5-HT, on cytoplasmic Ca 2+ levels in early sea urchin embryos. Effects of 5-HT antagonists and agonists during the first cleavage division TIC methiodide (PPM antagonist of 5HT 3 -receptors) applied during the first cleavage division (when cleavage furrow formation has already started) in Fura-2-dextran-ratio imaging experiments, evoked a Ca 2+ -rise in a dose-dependent manner (total of 41 experiments, Table 1, Fig. 1). DMSO (0.5%), used as a solvent of neurochemicals, had no significant effect (Table 1). Using the confocal microscope, the increase of free intracellular Ca 2+ caused by IM methiodide (100 μM) was observed in all of 6 experiments (intensity comparing to resting level increased by 45±6.4% with a latent period of 30 sec) (Fig. 2a,b). By comparison with the data from ratio imaging experiments, the rise in Ca 2+ -level corresponds to approximately 0.27±0.04 Short Contribution Abbreviations used in this paper: 5-HT, 5-hydroxytryptamine, serotonin; EDTA, ethylenediaminetetraacetic acid disodium salt; ASW, artificial sea water; BAPTA/AM, 1,2-bis (2-Aminophenoxy) ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester; Ca 2+ (i) , intracellular level of free calcium ions; Fura-2/ DX, Fura-2 dextran; CG-1/DX, Calcium Green-1 dextran; PIPES, piperazine; N,N’, tetraacetic acid; D-600, methoxyverapamil; 5-HTQ, trimethylserotonin methiodide; TIC, 3-tropanyl-indole-3-carboxylate hydrochloride (tropisetron); TIC methiodide, 3-tropanyl-indole-3-carboxylate methiodide; IM, inmecarb hydrochloride; IM methiodide, inmecarb methiodide; DMSO, dimethylsulfoxide; EPM, substance, easily penetrating the cell membrane; PPM, substance, poorly penetrating the cell membrane. μM. The duration of intracellular free Ca2+ elevation was from 1.5 to 7 min. Similar results were obtained in experiments with KYuR-14 methiodide (PPM 5-HT-antagonist, 75 μM, 3 experiments. Specificity of the effects of 5-HT-antagonists 5-HTQ (PPM 5HT 3 -agonist, 100 μM) administered 10-40 sec before TIC methiodide (100 μM) significantly decreased the effect of the latter in Fura-2-Dextran ratio imaging experiments (Table 2, Fig. 3), but only areas under the peaks differed significantly. Preliminary data show that the protective action of 5-HT (100 μM) was weaker than 5-HTQ. In confocal microscope experiments, 5-HTQ (100 μM) administered 1 min before IM methiodide (100 μM) completely prevented Ca 2+ increase in 2 out of 5 experiments, and left it in more or less unchanged form in another 3 experiments. No significant effects of 5-HTQ (100 μM) itself were observed.